Pharmacognosy Studies of Waltheria Indica L. of the Family Sterculiaceae

AEGAEUM JOURNAL ISSN NO: 0776-3808

Pharmacognosy Studies of Waltheria indica L. of the Family Sterculiaceae

Dr. Ch. Subbalakshmi 1 & * Dr. G. Meerabai2

1Research Scholar, Department of Botany,

Sri Krishnadevaraya University, Ananthapur, A.P., India.

2Assistant Professor, Department of Botany,

Rayalaseema University, Kurnool, A.P., India.

1Email : sivakesavulu [email protected]

2Email: [email protected]

Abstract: All plants produce chemical compounds as part of their normal metabolic activities, and they synthesize a curious variety of phyto chemicals. Some of the bioactive substances that can be derived from plants are flavonoids, alkaloids, carotenoids, tannin, antioxidants and phenolic compounds. They are all having medicinal importance and used as crude drugs in traditional medicine. The knowledge of the action of a drug can be utilized successfully only when the identity, physical nature and chemical constituents of the drug are well known, and pharmacognosy supplies this information. Pharmacognostic studies ensure endorsement of the plants and reproducible quality of herbal products which will shove to wellbeing and effectiveness of natural products. Pharmacognostic parameters include determination of number of stomata (mm-2), stomatal index, total number of epidermal cells (mm-2) and type of trichomes in leaf. Physicochemical studies like ash analysis and moisture contents, fluorescence analysis, extractive values and inorganic mineral analysis verify the identity of plant, the quality and purity of crude drug,

Therefore the morphology, macroscopic, microscopic, physical and physiochemical studies of Waltheria indica L. of the family Sterculiaceae was carried out in order to know its Pharmacognostic importance. Key words: Waltheria indica, Pharmacognosy, morphology, physical characters, crude drugs and physico chemical.

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1. Introduction: The Indian subcontinent is a vast repository of medicinal plants that are used in traditional medical treatments. Use of plants as a source of medicine has been an ancient practice and is an important component of the health care system in India. Interest in traditional medicines is growing rapidly due to the attention being given to it by the governmental agencies and different NGO comprises of general public and researchers as well as the increased side effects, adverse drug reactions and cost factor of the modern medicines. Pharmacognosy is "the study of the physical, chemical, biochemical, and biological properties of drugs, drug substances, or potential drugs or drug substances of natural origin as well as the search for new drugs from natural sources". The systematic study of natural medicines in terms of purity, potency, consistency and safety has become the major issues in Pharmacognosy. It deals with the collection, identification, preparation, and extraction of a large group of drugs obtained from natural sources, which are used both in orthodox and traditional medicine.

Waltheria indica, L. of the family Sterculiaceae is commonly used in traditional medicine against pain, inflammation, diarrhoea, dysentery, conjunctivitis, wounds, abscess, epilepsy, convulsions, anaemia, bladder ailments, astringent, febrifuge, abortifacient and asthma. Therefore the morphology, macroscopic, microscopic, physical and physiochemical studies of W. indica L. was carried out in order to know its Pharmacognostic importance.

2. Materials & Methods: 2.1. Epidermal Studies: The present work deals with the foliar epidermal morphology. Mature fresh leaves or dried leaves from herbarium were used to prepare the epidermal peels. Several techniques were followed for the preparation of epidermal peels and clearing of leaf bits depending upon the texture of leaves. Dried leaves were softened by immersing in water for 24 to 48 hours and they were of same age and size. The leaves were cut into suitable pieces and placed down on a microscopic slide and soaked in a few drops of 5% sodium hypochlorite. One end is held firmly with thumb and the other end scraped gently with a safety razor blade. If necessary extra hypochlorite was added, so that finally a thin clear area was cut off. The piece was placed in a cavity block for a few minutes in sodium hypochlorite and then washed in water filled in a petridish. Then loosely adhering cells were brushed off with the help of pointed hair brush.

In case of herbarium material, the leaves were boiled in water for about of 5-1 minutes. The chemical method was followed for the separation of the peels. Diluted nitric acid and chromic acid (5-10%) were used in different proportions. In some cases using three acid treatment (TAT) methods [19] was followed. Epidermal peels were stained in safranin (1%) and mounted in glycerin and made semi-permanent slides by ringing with nail paints. In case of exceptionally hairy leaves, the hairs were removed prior to separation of epidermal peels by covering the leaf surface with stick fast and gently peeling off the gum dried.

Epidermal peels which were obtained by these methods were stained with aqueous safranine and mounted on a clear microscopic slide using glycerine and sealed with DPX- mountant. The preparations were examined under microscope and photographs taken by using camera at 40×10x,15×10x,10×10x and 5×10x magnification.

The quantitative values of epidermis such as size of epidermal cells ( S.E.S), size of stomata (S.S), epidermal cell frequency/ mm2(E.C.F), stomatal frequency/mm2(S.F), percentage of stomatal types and length and breadth of trichomes on both the surfaces were recorded. For calculating ECF, the epidermal cells as well as stomatal subsidiaries were also counted excluding costal cells. The values are average of derived usually from readings. Stomatal Index is calculated for both the surfaces in case of amphistomatic leaves and abaxial in case of

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hypostomatic leaves. Stomatal index was calculated using the formula of Salisbury’s ,1927 [8] method.

Where ‘S’ denotes number of stomata per unit area and E number of epidermal cells of the same area. Measurement of the epidermal cell width was taken at the widest point on each cell. Trichome size (length and breadth) is measured in µm. The range of length and mean is recorded. Number of trichomes per square millimetre is calculated on both surfaces.

2.2. Physico-chemical Constants: The procedures recommended in Indian Pharmacopoeia and WHO guidelines were followed to calculate the physico-chemical constants. Physico- chemical parameters such as colour, consistency, pH and percent yield (% w/w). were determined for all plant extracts. 2.2.1. Ash values: Total ash value: The total ash was determined by incinerating 2-3gms. of accurately weighed air dried coarsely powdered drug in a tarred silica crucible which was previously ignited and cooled before weighing, at a temperature not exceeding 4500C. The ignition was repeated and the percentage of ash with reference to air-dried drug was calculated. Water soluble ash: The total ash was boiled for 5min with 25 ml of water. The residue was washed with hot water, ignited for 15min at a temperature not exceeding 4500C, cooled and weighed. This weight was subtracted from the weight of ash, the difference in weight represents the water soluble ash. The percentage of water soluble ash was calculated with reference to air- dried drug. Acid insoluble ash: The ash obtained was boiled with 25 ml of dilute hydrochloric acid for 5min. and filtered through an ash less filter paper. The residue was washed with hot water, ignited, cooled in a desiccator and weighed. The percentage of acid insoluble ash was calculated with reference to air dried drug. Sulphated ash: The sulphated ash was determined by incinerating 1 gm of accurately weighed air dried coarsely powdered drug in a tarred silica crucible which was previously ignited and cooled before weighing at a temperature not exceeding 4500C. The residue was moistened with 1 ml of concentrated sulphuric acid, ignited at 800±250C until all black particles have disappeared. It was then cooled; again sulphuric acid was added and ignited. It was cooled and the percentage of sulphated ash was calculated with reference to air dried drug. The preliminary phytochemical investigations were conducted employing various phytochemical tests and the presence of various phytochemical constituents was detected. 2.2.2. Extractive values: Ethanol soluble extractive: 5gms. of dried coarse powder of plants were macerated with 100ml of 90% ethanol in a closed flask for 24hrs, shaken frequently during 6 hours and allowed to stand for 18hrs. Filtered immediately taking precautions against loss of ethanol. 25ml of the filtrate was evaporated to dryness in a tarred flat bottomed shallow dish. The residue was dried at 1050C and weighed. The percentage of ethanol soluble extractive was calculated with reference to air dried drug.

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Water soluble extractive: 5gms of coarse powder was weighed and dissolved in 100ml of water in a stoppered flask, heated at 800C, shaken well and allowed to stand for 10min. It was cooled. 2gms of kieselghur was added and filtered. 5ml of the filtrate was transferred to a tarred evaporating dish and the solvent was evaporated on a water bath. The percentage of water soluble extractive was calculated with reference to air dried drug. 2.2.3. Determination of volatile oil in drug: 50gms of the drug was boiled with water in a Clavenger’s apparatus. The process was continued till no more oil was collected in the graduated tube. The volume of oil was measured and expressed in percentage. 2.2.4. Determination of crude fibre content: About 2gms of the drug was accurately weighed and extracted with ether. Then 200ml of 1.25% sulphuric acid was added and boiled for 30min under reflux. It was filtered and washed with boiling water until free of acid. The entire residue was rinsed back into flask with 200ml of boiling 1.25% sodium hydroxide solution and again boiled under reflux for 30min. The liquid was quickly filtered and the residue was washed with boiling water until neutral, dried at 1100C to constant weight. It was then ignited to 30min at 6000C, cooled and weighed. The percentage of crude fibre content was calculated with reference to the air dried drug. 2.2.5. Determination of loss on drying: Glass stoppered shallow bottle was weighed that had been dried in the same conditions to be employed in the determination. About 1gm of the sample was transferred to the bottle and distributed evenly by gently side wise shaking to a depth not exceeding 10mm. Place the loaded bottle in a drying chamber (the stopper was removed and left in the chamber). The sample was dried to a constant weight and allowed to cool. The bottle along with the content was weighed. The process was repeated until the successive weights differed not more than 0.5mg (drying to constant weight). The percentage loss of weight was calculated with reference to the air dried drug. 2.2.6. Determination of foaming Index: 1gm of the coarsely powdered drug was weighed and transferred to 500ml conical flask containing 100ml of boiling water. The flask was maintained at moderate boiling at 80-900C for about 30min. It was cooled, filtered into a volumetric flask and sufficient water was added through the filter to make up the volume to 100ml. Ten stoppered test tubes were cleaned (height 16cm, diameter 1- 6cm) and marked from 1 to 10. 1, 2, 3ml up to 10ml of the filtrate was measured and transferred to each tube and adjusted the volume of the liquid with water to 10ml. Then the tubes were stoppered and shaken lengthwise for 15sec uniformly, allowed to stand for 15min the length of the foam was measured in each tube. If the height of the foam in each tube is more than 1cm, the foaming index is more than 1000. In this case, 10ml of the first decoction of the plant material is measured and transferred to a 100ml volumetric flask (V2) and the volume is made to 100ml and followed the same procedure. 2.2.7. Fluorescence Analysis: The fluorescence analysis of the drug powder as well as various extracts was carried out by using the method of Chase and Pratt. The behaviour of the powder with different chemical reagents was also carried out. 2.2.8. Inorganic Mineral Analysis: Chemical analysis of higher plants in general has revealed the presence of 40 or more elements. Plant physiologists have proved that 18 of these elements are indispensable to plants and human beings require 28 or more elements. Of these elements, carbon, hydrogen, oxygen and nitrogen are present in larger quantities than others. Sulphur and phosphorous are present in protoplasm and has constituents of proteins or other important organic compounds. A study of inorganic constituents of plants is of interest to research workers in several fields, such as nutrition medicine and others because plants constitute direct or indirect sources of many of the elements, which are essential to animals including man. Therefore, the plant material was subjected to inorganic mineral analysis. Preparation of sample solution for inorganic mineral analysis: The plant material (10gms) was digested with 10 ml of nitric acid and left over night. It was then heated on a hot plate until

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the reddish brown colour ceased and cooled. A small volume of perchloric acid was added and transferred to a 50ml standard flask and made up to volume with double distilled water. 2.2.9. Determination of sodium and potassium by flame photometry: Flame photometry is based on the measurement of intensity of light emitted when a metal is introduced into the flame. The material when introduced into the flame is converted into gaseous state. The gaseous molecules are progressively dissociated to give free neutral atoms or radicals, which are excited by thermal energy of the flame. The excited atoms which are unstable quickly emits photons and return to the lower energy state, evenly reaching the unexcited state. The measurement of the emitted photons forms the basis of flame photometry. A plot of emission versus concentration in micrograms is prepared. The given unknown solution was diluted suitably and aspirated into the instrument. From the emission intensity produced, concentration of metal is determined by the interpolation of calibration curve. The instrument used was Systronics Flame Photometer. Procedure: A series of standard solutions containing the element to be determined in increasing concentrations within the concentration range recommended for the instrument were prepared. Nitric acid and perchloric acid used for the preparation of sample solution of the plant material were also added in the same concentration to the standard solution. The appropriate filter was chosen, water was sprayed into the flame and the galvanometer reading was adjusted to zero. The most concentrated solution was then sprayed into the flame and the galvanometer reading was recorded. Again, water was sprayed till the galvanometer reading was zero. Then the standard solution was sprayed into the flame and the procedure was repeated three times for each concentration. A calibration curve was prepared by plotting the mean of three readings of each standard against the concentration. The sample solution prepared as above was then aspirated into the flame three times, the galvanometer reading was recorded and the apparatus was washed thoroughly with water after each aspiration. Using the mean of three readings, the concentration of the element being examined was determined from the calibration curve. To confirm the concentration thus obtained, the operation was repeated with the standard solution of the same concentration as that of the solution being examined. 2.2.10. Determination of calcium, cobalt, iron, copper, magnesium and manganese by atomic absorption spectroscopy: This technique is based on the fact that when atoms, ions or ion complexes of an element in the ground state are atomized in a flame, the absorbed light has the characteristic wavelength of that element. If the absorption process takes place under reproducible conditions the absorption is proportional to the number of absorbing atoms. The concentration of the metal is determined by interpolation of the calibration curve. The instrument used was Perkin Elmer Atomic Absorption Spectrophotometer. Procedure: Three standard solutions of the element to be determined covering the concentration range recommended for the instrument were prepared. Nitric acid and perchloric acid used in the preparation of the substance being examined were also added to the standard solutions in the same concentration. After calibration of the instrument, each standard solution was introduced into the flame three times and the steady reading was recorded. The apparatus was thoroughly washed after each introduction. A calibration curve was prepared by plotting the mean of each group of three readings against concentration. The plant extract prepared above was then introduced into the flame and the reading was recorded. The sequence was then repeated twice. Using the mean of the three readings, the concentration of the element was determined from the calibration curve. The process was repeated for the determination of other elements using different lamps. 3.Results & Discussion: 3.1.Adaxial surface of lamina: Epidermal cell complex: Epidermal cells: Epidermal cells polygonal, 5-6-sided, some are rectangular iso or aniso diametric, anticlinal walls sinuate, moderately thick, contents scanty, surface smooth,

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distributed all over, variously oriented. E.C.F: 676 (44)/ mm2, S.E.C: 52 (6) × 24 (3) µm. Costal cell: 4-sided, linear, elongated, narrow, walls thin, contents scanty, surface smooth, occur in mid vein and lateral veins. Stomatal complex: Stomata mostly aniso cytic, anomo cytic also occur, multicyclic, elliptical-shaped, subsidiary cells indistinct, normally of a- and f-type, variously contiguous stomata occur. Distributed all over and variously oriented. S.F: 204 (19)/ mm2, S.I., 23.18, S.S. 21 (1.8) × 17 (1.8) µm. Trichome complex: Uniseriate, macroform, stellate hair: Foot: 1-celled, contents scanty, walls thin. Stalk: uniseriate, cylindrical, 3-10-celled in length, cells of varied lengths, broader than long or vice-versa. Contents dense, walls thin, surface smooth. Head: unicellular, stellate, 7- armed, some arms dichotomously divided, each arm tapering towards the tip, contents scanty, walls thick, surface smooth. 3.2. Abaxial surface of lamina: Epidermal cell complex: Epidermal cells: Epidermal cells polygonal, 5-6-sided, some are rectangular iso or anisodiametric, anticlinal walls sinuate, moderately thick, contents scanty, surface smooth, distributed all over, variously oriented. E.C.F: 796 (24) mm2, S.E.C: 51(4) × 23(4) µm. Costal Cells: 4-sided, linear, elongated, narrow, walls thin, contents scanty, surface smooth, occur in mid vein and lateral veins. Stomatal complex: As on adxial surface of lamina, but the subsidiaries are of a-type and c- type. S.F: 233(18)/mm2, S.I: 23.64: S.S.: 27 × 19 µm. Trichome complex: Uniseriate macroform stellate hair: Foot: 1-celled, contents scanty, wall thin. Stalk: unseriate, cylindrical, 3-10-celled in length, cells of varied lengths, broader than long or vice-versa. Contents dense, walls thin, surface smooth. Head: unicellular, stellate, 6-7- armed, some arms dichotomously divided, each arm tapering towards the tip, contents scanty, walls thick, surface smooth. The present work brings out the epidermal morphology of mature leaves. The present observations show that in W. indica cell wall shape is sinuate (irregular) and confirm earlier observations made by Stace, 1965 [3]. The present observations show that the species of W. indica cell wall shape polygonal and confirm earlier observations made by Aworinde et al. , 2012 [6]. Stomata were observed to be variously oriented in leaves especially in those with reticulate venation where as they were parallel oriented in leaves with parallel venation [11]. In the present study stomata are distributed irregularly with their long axis facing all directions. This is correlated with the reticulate venation of the leaves. According to Stace, 1965 [3], the distribution and frequency of stomata are of considerable taxonomic value. Striking differences regarding the numerical frequency of the stomata have been shown in a number of plants growing in different environmental conditions [8], in the leaves produced at different height of the plant, in different size of the leaves and also from one of the surfaces of the leaf to the other [12]. Such difference has been observed in Waltheria indica. Aworinde et al., 2012[6] reported that stomata are amphistomatic and anisocytic type. This is confirmed in the present study also. In the plant epidermis, trichome complex represents probably the most interesting element due to diversity in structure and distribution. The trichomes are frequently present, easily observable and show variation [9;5;10]. They are used as tool of identification of plants from ordinal rank to specific level [5;17;18;21;25;26;20;27;28]. The length of the trichomes and frequency of the trichome vary from species to species and type to type.

With help of the above foliar epidermal characters combined with the statistical data like epidermal cell frequency, Stomatal frequency, Stomatal Index and Trichome features found to be valuable to prepare a taxonomic key for the identification of the species studied.

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3.3. Physico chemical characteristics of the crude drug Ash analysis and moisture contents: In the present study ash analysis for crude powder drug of leaf was carried out. It was observed, the total ash was (6.8%) , acid insoluble ash was 4.27% water soluble ash was 2.34 % and sulphated ash was 6.3%. The crude fiber content was 17.32%. The results are given in table -1. Table – 1 ; Physico-chemical constants of Waltheria indica L.

S .No. Parameters Percentage (%w/w) 1. Total ash 6.8 2. Acid insoluble ash 4.27 3. Water soluble ash 2.34 4. Sulphated ash 6.3 5. Solubility Water soluble extractive 12.6 Alcohol soluble extractive 2.1 6. Crude fibre content 17.32 7. Loss on drying 4.39 8. Foaming index > 100 3.4. Fluorescence Analysis: Fluorescence study with UV light is a very useful tool for evaluation of crude drugs. Crude extract or powder drugs, when viewed under UV light give characteristic fluorescence [29]. The fluorescence phenomenon is the emission of light of different wave lengths, under the influence of UV light, due to different chemical constituents, otherwise not observable in ordinary visible light [13;7;14]. Fluorescence study helps for authentication and standardization of crude drugs. Saveral Crud drugs of plant origin have been authenticated though the fluorescence study technique e.g., Morinda umbellata [30], Holoptelea integrifolia [23], root and stem of Ichnocarpus frutescens [2], leaves of Catunaregum spinosa [24], Hygrophila auriculata [16] and Crocus sativus [32]. Powder drug leaf was such and after treatment with various solvents was subjected to fluorescence analysis. Observations were made under visible light and under UV light of short wave length and long wave length. The fluorescence analysis of powder with various reagents and extracts were given in Table 2. Fluorescence study is very useful, quick, easy and unfailing method for the detection of adulterations. 3.5. Extractive values: Extractions of crude powder drug with different solvents gives different extractive values. Extractive value is one of the useful methods for evaluation of crude drugs, and provides guidance about the most suitable solvent to be used for extraction, and also helps in detecting various types of adulteration and exhausted materials i.e., water and alcohol soluble extractive values can be used for the detection of adulterants, defective processing and poor quality of the drug while petroleum ether soluble extractive value indicates lipid contents present in the crude drugs [31;4]. Table – 2; Fluorescence analysis of powder of W. Indica L. S.No Reagents Day light Short UV Long UV (365 nm) 1. Powdered plant Green Dark Green Green 2. Powder + 1 N HCl Yellow Green Pale Green 3. Powder + 1 N NaOH Light Red Light Green Dark Green 4. Powder + 50% HCl Yellow Fluorescent Green Green 5. Powder + 50% H2SO4 Green Light Green Green 6. Powder +50%HNO3 Brown Pale Brown Brown 7. Powder + Methanol Fluorescent Light Green Green Green 8. Powder + Methanol + 1 N NaOH Green Brown Pale Green

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Fluorescence analysis of various extracts

S.No Extracts Day light UV light Short (254nm) Long (365 nm) 1. n-Hexane Dark green Green Dark green 2. Chloroform Dark brown Pale green Dark green 3. Ethyl acetate Dark green Green Light green 4. Methanol Green Green Green

3.6.Inorganic Mineral Analysis: The amount of sodium present in plant material was estimated by flame photometry. The amount of other metals present was estimated by Atomic absorption spectroscopy and the results are given in Table 3. The study revealed the presence of high iron content (864mg/kg) in leaves. Table – 3; Inorganic mineral analysis of Waltheria indica

Parameters Amount present (mg/kg) S.No 1. Total Iron 864 2. Cadmium (DB) < 0.0003 3. Copper (DB) 86 4. Chromium (DB) < 0.02 5. Cyanide (DB) < 0.001 6. Cobalt (DB) < 0.003 7. Lead (DB) < 0.025 (DB-Dry basis 8. Manganese (DB) 539 WB –Wet basis) 9. Nickel (DB) < 0.002 10. Zinc (DB) 87 11. Sodium (WB) 210 12. Total phosphate (DB) 218

Plants have the ability to accumulate such essential elements in their different parts, which are of highest importance in human nutrition [22]. There are also some toxic heavy metals like Co, Cd etc., which are not directly required by the plants, but still they are accumulated in some plants due to environmental pollutions [15] which create serious health hazards, when the plants are consumed by the humans and animals [33]. . In present study, toxic metals were reported in very meagre quantities. In human body, trace elements play a vital role both in prevention and treatment of different human diseases [1]. 4.Conclusion:The quantitative determinations of some pharmacognostic parameters are useful for setting standards for crude drugs. Since plants investigated are useful in the traditional medicine for the treatment of some ailments, it is important to standardize them for use as drugs. The pharmacognostic constants for the leaves of plants, the diagnostic microscopic features and the numerical standards reported in this work could be useful for the compilation of suitable monographs for their proper identification. 5. References: 1. A.E.M. Saeed, and R.S.M. Bashir, “Physiochemical analysis of Ximenia americana L. seed oil and Structure elucidation of some chemical constitution of its seed oil and fruit pulp”. J. Pharmco. & Phytoth. Vol. 2, no.4, (2010). PP. 49-55. 2. C. Kalidass, D.A. Abragam and V.R. Mohan, “Pharmacognostic studies on Ichnocarpus frutescens (L.) R.Br”. J. Herbal Medicine and Toxicol. Vol. 3, no.2: (2009.), pp. 9-15. 3. C.A. Stace, “Cuticular studies as an aid to plant taxonomy”. Bull. Mus. Nat. Hist., 4: (1965). Pp. 1-78. 4. C. K. Kokate, “Practical Pharmacognosy”.4th Ed. Vallabh Prakashan. New Delhi. 5. C.R. Metcalfe and L. Chalk, “Anatomy of the Dicotyledons,” Vol. I and II. Clarendon Press, Oxford. (1950). 6. D.O. Aworinde, B.O., Ogundairo, K.F., Osuntoyinbo, and O.A. Olanloye, “Foliar epidermal characters of some Sterculiaceae species in Nigeria”. Bayero Journal of Pure and Applied Sciences. Vol. 5, no. 1. (2012). Pp.48-56. 7. E.E. Jarald, and S.E. Jarald, “A text book of pharmacognosy and phytochemistry”. CBS Publisher and distributers, New Delhi. (2007).

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